Activities

Activity 9.1: Restriction Digestion of DNA (In Silico and Lab Protocol)

Aim: To perform a restriction digestion of Lambda DNA using EcoRI and HindIII.

Materials Required:

Lambda DNA (0.5 $\mu$ g/ $\mu$ L)
Restriction Enzymes: EcoRI and HindIII (10 units/ $\mu$ L)
10X Assay Buffer (specific to the enzyme, e.g., Tris-HCl, $MgCl_2$ , $NaCl$ , DTT)
Nuclease-free water (DEPC-treated)
Microcentrifuge tubes (0.5 mL)
Micropipettes (0.5 - 10 $\mu$ L range)
Water bath or Dry bath (set to 37°C)

Procedure:

Set up the reaction mixture in a microcentrifuge tube on ice:
- Lambda DNA: 2.0 $\mu$ L (1 $\mu$ g)
- 10X Assay Buffer: 2.0 $\mu$ L
- Restriction Enzyme (EcoRI): 1.0 $\mu$ L (10 Units)
- Nuclease-free water: 15.0 $\mu$ L
- Total Volume: 20.0 $\mu$ L
Mix by gentle flicking and pulse-spin in a microcentrifuge for 5 seconds.
Incubate the tubes at 37°C for exactly 1 hour.
Stop the reaction by adding 4 $\mu$ L of 6X DNA Loading Dye and heating at 65°C for 10 minutes (to denature the enzyme).

Scientific Note One unit (U) of restriction enzyme is defined as the amount required to digest 1 $\mu$ g of Lambda DNA in 1 hour at 37°C in a 50 $\mu$ L reaction volume. EcoRI recognizes the palindromic sequence 5'-GAATTC-3' and cuts between G and A, creating 4-base 5' overhangs (sticky ends).

Lab Best Practice Always add the enzyme last. Restriction enzymes are stored in 50% Glycerol to prevent freezing at -20°C; however, Glycerol concentration in the final reaction should not exceed 5% to prevent "Star Activity" (non-specific cutting).

Activity 9.2: Agarose Gel Electrophoresis

Aim: To separate DNA fragments based on their molecular weight.

Materials Required:

Molecular biology grade Agarose
1X TAE Buffer (Tris-Acetate-EDTA, pH 8.0)
Ethidium Bromide ( $EtBr$ , 10 mg/mL) or SYBR Safe
DNA Ladder (1 kb or 100 bp)
Electrophoresis tank and power supply
Gel casting tray and combs

Procedure:

Gel Preparation: Weigh 0.8g of agarose and add to 100 mL of 1X TAE buffer (for a 0.8% gel).
Heat in a microwave until the agarose is completely dissolved and the solution is clear.
Cool to approx. 55°C, then add 5 $\mu$ L of Ethidium Bromide.
Pour the gel into the casting tray with the comb in place. Allow it to solidify for 30 minutes.
Loading: Remove the comb, place the gel in the tank, and submerge in 1X TAE buffer.
Load 10 $\mu$ L of the digested DNA sample (from Activity 9.1) into the wells.
Run the electrophoresis at 100V for 45-60 minutes.

Safety First Ethidium Bromide is a potent mutagen and a possible carcinogen. Always wear gloves, a lab coat, and safety goggles. Use a UV transilluminator with a protective shield to visualize the DNA bands.

Scientific Note DNA is negatively charged due to the phosphate backbone. In an electric field, it migrates towards the positive anode (+). The agarose matrix acts as a molecular sieve; the migration distance is inversely proportional to the $log_{10}$ of the molecular weight of the DNA fragment.

Activity 9.3: Polymerase Chain Reaction (PCR) - Temperature Profile

Aim: To amplify a specific DNA segment using Taq DNA polymerase.

Materials Required:

Template DNA (10-50 ng)
Forward and Reverse Primers (10 $\mu$ M each)
dNTP Mix (10 mM each of dATP, dCTP, dGTP, dTTP)
10X PCR Buffer (with $MgCl_2$ )
Taq DNA Polymerase (5 U/ $\mu$ L)
Thermal Cycler (PCR Machine)

Procedure:

Set up a 25 $\mu$ L reaction on ice.
Program the thermal cycler with the following 30-cycle profile:
- Initial Denaturation: 95°C for 2 minutes (1 cycle)
- Denaturation: 95°C for 30 seconds
- Annealing: 55°C (depends on Primer Tm) for 30 seconds
- Extension: 72°C for 1 minute per kb of product
- Final Extension: 72°C for 10 minutes (1 cycle)
- Hold: 4°C indefinitely.

Lab Best Practice The annealing temperature ( $T_a$ ) is usually set 5°C below the melting temperature ( $T_m$ ) of the primers. If $T_a$ is too low, non-specific amplification occurs; if too high, no product is formed.

Scientific Note Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, has an optimum activity temperature of 72°C-75°C and remains stable even at 95°C, which is necessary for the denaturation of the DNA template in every cycle.

Activities

Activity 9.1: Restriction Digestion of DNA (In Silico and Lab Protocol)

Aim: To perform a restriction digestion of Lambda DNA using EcoRI and HindIII.

Materials Required:

Lambda DNA (0.5 $\mu$ g/ $\mu$ L)
Restriction Enzymes: EcoRI and HindIII (10 units/ $\mu$ L)
10X Assay Buffer (specific to the enzyme, e.g., Tris-HCl, $MgCl_2$ , $NaCl$ , DTT)
Nuclease-free water (DEPC-treated)
Microcentrifuge tubes (0.5 mL)
Micropipettes (0.5 - 10 $\mu$ L range)
Water bath or Dry bath (set to 37°C)

Procedure:

Set up the reaction mixture in a microcentrifuge tube on ice:
- Lambda DNA: 2.0 $\mu$ L (1 $\mu$ g)
- 10X Assay Buffer: 2.0 $\mu$ L
- Restriction Enzyme (EcoRI): 1.0 $\mu$ L (10 Units)
- Nuclease-free water: 15.0 $\mu$ L
- Total Volume: 20.0 $\mu$ L
Mix by gentle flicking and pulse-spin in a microcentrifuge for 5 seconds.
Incubate the tubes at 37°C for exactly 1 hour.
Stop the reaction by adding 4 $\mu$ L of 6X DNA Loading Dye and heating at 65°C for 10 minutes (to denature the enzyme).

Activity 9.2: Agarose Gel Electrophoresis

Aim: To separate DNA fragments based on their molecular weight.

Materials Required:

Molecular biology grade Agarose
1X TAE Buffer (Tris-Acetate-EDTA, pH 8.0)
Ethidium Bromide ( $EtBr$ , 10 mg/mL) or SYBR Safe
DNA Ladder (1 kb or 100 bp)
Electrophoresis tank and power supply
Gel casting tray and combs

Procedure:

Gel Preparation: Weigh 0.8g of agarose and add to 100 mL of 1X TAE buffer (for a 0.8% gel).
Heat in a microwave until the agarose is completely dissolved and the solution is clear.
Cool to approx. 55°C, then add 5 $\mu$ L of Ethidium Bromide.
Pour the gel into the casting tray with the comb in place. Allow it to solidify for 30 minutes.
Loading: Remove the comb, place the gel in the tank, and submerge in 1X TAE buffer.
Load 10 $\mu$ L of the digested DNA sample (from Activity 9.1) into the wells.
Run the electrophoresis at 100V for 45-60 minutes.

Activity 9.3: Polymerase Chain Reaction (PCR) - Temperature Profile

Aim: To amplify a specific DNA segment using Taq DNA polymerase.

Materials Required:

Template DNA (10-50 ng)
Forward and Reverse Primers (10 $\mu$ M each)
dNTP Mix (10 mM each of dATP, dCTP, dGTP, dTTP)
10X PCR Buffer (with $MgCl_2$ )
Taq DNA Polymerase (5 U/ $\mu$ L)
Thermal Cycler (PCR Machine)

Procedure:

Set up a 25 $\mu$ L reaction on ice.
Program the thermal cycler with the following 30-cycle profile:
- Initial Denaturation: 95°C for 2 minutes (1 cycle)
- Denaturation: 95°C for 30 seconds
- Annealing: 55°C (depends on Primer Tm) for 30 seconds
- Extension: 72°C for 1 minute per kb of product
- Final Extension: 72°C for 10 minutes (1 cycle)
- Hold: 4°C indefinitely.

Biotechnology: Principles and Processes - Activities

Activities

Activity 9.1: Restriction Digestion of DNA (In Silico and Lab Protocol)

Activity 9.2: Agarose Gel Electrophoresis

Activity 9.3: Polymerase Chain Reaction (PCR) - Temperature Profile

On this page

Biotechnology: Principles and Processes - Activities

Activities

Activity 9.1: Restriction Digestion of DNA (In Silico and Lab Protocol)

Activity 9.2: Agarose Gel Electrophoresis

Activity 9.3: Polymerase Chain Reaction (PCR) - Temperature Profile

On this page